U.S. patent application number 12/162566 was filed with the patent office on 2009-07-02 for non-coiled protective regions of pneumococcal surface proteins pspa and pspc.
This patent application is currently assigned to THE UNIVERSITY OF ALABAMA RESEARCH FOUNDATION. Invention is credited to David E. Briles, Susan K. Hollingshead.
Application Number | 20090170162 12/162566 |
Document ID | / |
Family ID | 38328037 |
Filed Date | 2009-07-02 |
United States Patent
Application |
20090170162 |
Kind Code |
A1 |
Hollingshead; Susan K. ; et
al. |
July 2, 2009 |
NON-COILED PROTECTIVE REGIONS OF PNEUMOCOCCAL SURFACE PROTEINS PSPA
AND PSPC
Abstract
The embodiments described herein provide for immunogenic
portions of Streptococcus pneumoniae surface protein A and surface
protein C lacking alpha helical structure.
Inventors: |
Hollingshead; Susan K.;
(Birmingham, AL) ; Briles; David E.; (Birmingham,
AL) |
Correspondence
Address: |
NIXON PEABODY, LLP
401 9TH STREET, NW, SUITE 900
WASHINGTON
DC
20004-2128
US
|
Assignee: |
THE UNIVERSITY OF ALABAMA RESEARCH
FOUNDATION
Birmingham
AL
|
Family ID: |
38328037 |
Appl. No.: |
12/162566 |
Filed: |
February 1, 2007 |
PCT Filed: |
February 1, 2007 |
PCT NO: |
PCT/US2007/002698 |
371 Date: |
November 26, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60764731 |
Feb 2, 2006 |
|
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60851510 |
Oct 13, 2006 |
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Current U.S.
Class: |
435/69.3 ;
424/190.1; 435/320.1; 435/325; 530/300; 536/23.7 |
Current CPC
Class: |
A61K 2039/55505
20130101; A61P 37/02 20180101; A61P 27/06 20180101; C07K 14/3156
20130101; A61K 39/092 20130101; A61P 11/00 20180101; A61P 31/04
20180101; C07K 16/1275 20130101 |
Class at
Publication: |
435/69.3 ;
530/300; 536/23.7; 435/320.1; 435/325; 424/190.1 |
International
Class: |
C12P 21/02 20060101
C12P021/02; C07K 14/315 20060101 C07K014/315; C07H 21/02 20060101
C07H021/02; C12N 15/63 20060101 C12N015/63; C12N 5/10 20060101
C12N005/10; A61K 39/09 20060101 A61K039/09; A61P 37/02 20060101
A61P037/02 |
Goverment Interests
FEDERAL FUNDING
[0001] This invention was funded, in part, by the Federal
Government under NIH/NIAID Contract No. R01 AI21543. Accordingly,
the Federal Government may have certain rights in this invention.
Claims
1. A purified, immunogenic polypeptide of Streptococcus pneumoniae
surface protein A (PspA) or surface protein C (PspC) that lacks
alpha-helical structure.
2. A purified, immunogenic polypeptide having the amino acid
sequence of the non-proline block of a PspA or PspC molecule that
lacks alpha-helical structure.
3. A purified, immunogenic portion of Streptococcus pneumoniae
surface protein A (PspA) or surface protein C (PspC) having an
amino acid sequence selected from the group consisting of
TABLE-US-00030 [SEQ ID NO: 1]
DLKKAVNEPEKPAEEPENPAPAPKPAPAPQPEKPAPAPAPKPEKSADQQA
EEDYARRSEEEYNRLTQQQPPKAEKPAPAPVPKPEQPAPAPKTGWGQENG MW; [SEQ ID NO:
2] DLKKAVNEPETPAPAPAPAPAPAPTPEAPAPAPAPAPKPAPAPKPAPAPK
PAPAPKPAPAPKPAPAPKPAPAPAPAPKPEKPAEKPAPAPKPETPKTGWK QENGMW; [SEQ ID
NO: 3] MAKKAELEKTPEKPAEEPENPAPAPQPEKSADQQAEEDYARRSEEEYNRL TQQQPPKA;
[SEQ ID NO: 4] EKSADQQAEEDYARRSEEEYNRLTQQQ;
and immunogenic homologs, portions, fragments, variants, or
derivatives of any of the foregoing.
4. A vaccine comprising an immunogenic polypeptide of (PspA) or
(PspC) that lacks alpha-helical structure.
5. The vaccine of claim 4, in which said portion consists of a
polypeptide having the amino acid of SEQ ID NO. 1, NO: 2, NO: 3, or
NO: 4 or an immunogenic homolog, portion, fragment, variant, or
derivative thereof.
6. A purified nucleic acid that encodes an immunogenic
non-alpha-helical region of a PspA or PspC polypeptide.
7. The nucleic acid of claim 6 having a DNA sequence:
TABLE-US-00031 [SEQ ID NO: 10]
GACCTTAAGAAAGCAGTTAATGAGCCAGAAAAACCAGCTGAAGAGCCTGA
GAATCCAGCTCCTGCACCAAAACCAGCGCCGGCTCCTCAACCAGAAAAAC
CAGCTCCAGCTCCTGCACCAAAACCAGAGAAGTCAGCAGATCAACAAGCT
GAAGAAGACTATGCTCGTAGATCAGAAGAAGAATATAACCGCTTGACTCA
ACAGCAACCGCCAAAAGCAGAAAAACCAGCTCCAGCTCCTGTACCAAAAC
CAGAGCAACCAGCTCCCGCACCAAAAACGGGCTGGGGACAAGAAAACGGT ATGTGG; [SEQ ID
NO: 11] GACCTTAAGAAAGCAGTTAATGAGCCAGAAACTCCAGCTCCGGCTCCAGC
CCCAGCTCCAGCTCCAGCTCCAACTCCAGAAGCCCCAGCTCCAGCTCCAG
CTCCGGCTCCTAAACCAGCTCCGGCTCCTAAACCAGCTCCGGCTCCTAAA
CCAGCTCCGGCTCCTAAACCAGCTCCGGCTCCTAAACCAGCTCCGGCTCC
TAAACCAGCTCCAGCTCCAGCTCCGGCTCCTAAACCAGAAAAGCCAGCAG
AAAAACCAGCTCCAGCTCCTAAACCAGAAACTCCAAAAACAGGCTGGAAA
CAAGAAAACGGTATGTGG; [SEQ ID NO: 12
ATGGCTAAAAAAGCTGAATTAGAAAAAACTCCAGAAAAACCAGCTGAAGA
GCCTGAGAATCCAGCTCCAGCACCACAACCAGAGAAGTCAGCAGATCAAC
AAGCTGAAGAAGACTATGCTCGTAGATCAGAAGAAGAATATAATCGCTTG
ACCCAACAGCAACCGCCAAAAGCA; [SEQ ID NO: 13]
GAGAAGTCAGCAGATCAACAAGCTGAAGAAGACTATGCTCGTAGATCAGA
AGAAGAATATAATCGCTTGACCCAACAGCAACCG; or [SEQ ID NO: 14]
GAGAAGTCAGCAGATCAACAAGCTGAAGAAGACTATGCTCGTAGATCAGA
AGAAGAATATAACCGCTTGACTCAACAGCAACCG.
8. A vector comprising a nucleic acid of claim 6 or 7.
9. A host cell comprising the nucleic acid of claim 6.
10. A host cell comprising the vector of claims 8.
11. A method of making an immunogenic polypeptide constituting a
non-alpha-helical region of a PspA or PspC polypeptide comprising
the step of preparing the polypeptide from a host cell of claim 9
or claim 10 that produces said polypeptide.
12. A host cell capable of producing a poly peptide with the amino
acid sequence of claim 3.
13. A method of immunizing a patient comprising administering an
effective amount of at least one immunogenic polypeptide
constituting a non-alpha-helical region of a PspA or PspC
polypeptide.
14. A purified immunogenic polypeptide comprising the amino acid
sequence TABLE-US-00032 EKSADQQAEEDYARRSEEEYNRLTQQQ. [SEQ ID NO:
4]
15. The immunogenic polypeptide of claim 14 comprising the amino
acid sequence selected from the group consisting of TABLE-US-00033
[SEQ ID NO: 5] MEKSADQQAEEDYARRSEEEYNRLTQQQ; [SEQ ID NO: 8]
EKSADQQAEEDYARRSEEEYNRLTQQQP; [SEQ ID NO: 1]
DLKKAVNEPEKPAEEPENPAPAPKPAPAPQPEKPAPAPAPKPEKSADQQA
EEDYARRSEEEYNRLTQQQPPKAEKPAPAPVPKPEQPAPAPKTGWGQENG MW; and [SEQ ID
NO: 3] MAKKAELEKTPEKPAEEPENPAPAPQPEKSADQQAEEDYARRSEEEYNRL TQQQPPKA.
Description
FIELD OF THE INVENTION
[0002] The embodiments described herein relate to molecular
immunology, bacteriology, and vaccine development. More
specifically, the various embodiments relate to antigenic and
immunogenic portions of Streptococcus pneumoniae surface protein A
and surface protein C that lack alpha-helical structure.
BACKGROUND OF THE INVENTION
[0003] Streptococcus pneumoniae is a well known human pathogen and
a major etiologic agent for pneumonia, meningitis, otitis media as
well as sepsis, among primarily young children and older adults.
Antibodies to a capsular polysaccharide (PS) may provide protection
against pneumococci expressing the same capsular serotype.
Currently available pneumococcal vaccines contain a mixture of
capsular PS of multiple serotypes. For example, one pneumococcal
vaccine contains capsular PS from twenty-three commonly found
serotypes. The most recently developed type of vaccine contains
capsular PS from seven to thirteen serotypes that are conjugated to
a protein molecule. A seven-valent conjugate vaccine was introduced
in 2000 for clinical use in the USA, and has reduced the incidence
of invasive pneumococcal diseases in children and in adults.
[0004] An alternative approach for protecting children and the
elderly from pneumococcal infection employs protein antigens that
could elicit protective immune responses. Such proteins may serve
as a vaccine by themselves, may be used in conjunction with
successful polysaccharide-protein conjugates, or serve as carriers
for polysaccharide components. The pneumococcal surface protein A
(PspA) has been identified as an immunogenic protein with potential
for pneumococcal vaccines.
[0005] Most of the work concerning PspA as a potential for a
protein-based pneumococcal vaccine has focused on cross-protective
epitopes lying within the alpha-helical region of the PspA protein,
a region predicted to have a coiled-coil protein conformation. It
has been suggested, however, that this alpha-helical region may
have the potential to elicit antibodies that cross-react with
proteins of the human heart and skeletal muscles. On the other
hand, adults often have PspA antibodies, naturally elicited during
childhood, that have no connection to rheumatic heart disease or
auto-reactive immune syndromes. Nevertheless, there remains a need
for pneumococcal surface polypeptides that are immunogenic yet
minimize risk of self-reactive responses.
SUMMARY OF THE INVENTION
[0006] Embodiments described herein provide for non-alpha-helical
regions of PspA and PspC polypeptides that are capable of eliciting
an immune response. In some embodiments, these polypeptides, when
used as a component of a vaccine, provide protective immunity
against pneumococcal disease.
[0007] In other embodiments, the polypeptides have the amino acid
sequence DLKKAVNEPEKPAEEPENPAPAPKPAPAPQPEKPAPAPAPKPEKSADQQAEEDYARR
SEEEYNRLTQQQPPKAEKPAPAPVPKPEQPAPAPKTGWGQENGMW [SEQ ID NO: 1];
DLKKAVNEPETPAPAPAPAPAPAPTPEAPAPAPAPAPKPAPAPKPAPAPKPAPAPKPA
PAPKPAPAPKPAPAPAPAPKPEKPAEKPAPAPKPETPKTGWKQENGMW [SEQ ID NO: 2];
MAKKAELEKTPEKPAEEPENPAPAPQPEKSADQQAEEDYARRSEEEYNRLTQQQPPK A [SEQ ID
NO: 3]; or EKSADQQAEEDYARRSEEEYNRLTQQQ [SEQ ID NO: 4], and
antigenic or immunogenic homologs, portions, fragments, variants,
or derivatives of any of the foregoing.
[0008] Another aspect of the invention provides for a vaccine
comprising the non-alpha-helical regions of PspA and PspC
polypeptides. In other aspects, the vaccine may include one or more
of the peptides with the amino acid sequences designated in SEQ ID
NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 and antigenic or
immunogenic homologs, portions, fragments, variants, or derivatives
thereof.
[0009] Another embodiment provides for nucleic acids encoding the
immunogenic non-alpha-helical regions of PspA and PspC
polypeptides, vector comprising these nucleic acids, and host cells
comprising these nucleic acids or vectors. Another embodiment
provides for host cells that produce the immunogenic
non-alpha-helical regions of PspA and PspC polypeptides.
[0010] A further aspect provides for a method of making an
immunogenic polypeptide constituting a non-alpha-helical region of
a PspA or PspC polypeptide comprising the step of preparing the
polypeptide from a host cell that expresses the polypeptide.
[0011] Another aspect provides for a method of immunizing a patient
comprising administering an effective amount of at least one
immunogenic polypeptide constituting a non-alpha-helical region of
a PspA or PspC polypeptide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 depicts a map of the regions common to PspA (and in
some instances PspC) proteins of all known families and clades. It
also depicts recombinant Psp polypeptides MB001, PAC003, PAC001,
and NPB.
[0013] FIGS. 2A-E present the amino acid sequences of recombinant
PspA polypeptides.
[0014] FIGS. 3A-D present PspA-specific portions of embodiments of
recombinant protein immunogens.
[0015] FIG. 4 is a graphical representation of results of the mouse
sepsis challenge exemplified herein.
[0016] FIGS. 5A-C present Chou-Fasman structural probability plots
of the PspA-specific immunogenic polypeptides.
[0017] FIG. 6 shows the alignment of example embodiments of
immunogenic PspA/PspC polypeptides.
[0018] FIG. 7 presents data following immunization with
proline-rich regions of PspA and challenge with capsular type 3
strain 3JYP2670.
[0019] FIG. 8 shows a stained gel and corresponding Western blot
indicating that mAb KL67 is specific to the non-proline-block of
the proline-rich region of PspA/PspC polypeptides.
[0020] FIG. 9 presents data from fluorescent-activated cell sorting
(FACS) binding of mAb KL67 to several strains of pneumococci.
[0021] FIG. 10 presents data from FACS binding of mAb KL67 to WU2
wildtype strain and mutant strains that lack PspA and Hic, or
capsule.
[0022] FIG. 11 presents data from FACS binding of mAb KL67 to TIGR4
wild-type strain and mutant strains that lack capsule, PspA, PspC,
or both PspA and PspC.
[0023] FIGS. 12A-B indicate the similarity in the sequence of the
non-pro-block in the diverse pspA and pspC genes.
DETAILED DESCRIPTION OF THE INVENTION
[0024] It should be understood that this invention is not limited
to the particular methodology, protocols, and reagents, etc.,
described herein and as such may vary. The terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the present invention, which
is defined solely by the claims.
[0025] As used herein and in the claims, the singular forms "a,"
"an," and "the" include the plural reference unless the context
clearly indicates otherwise. Thus, for example, the reference to a
polypeptide is a reference to one or more such polypeptides,
including equivalents thereof known to those skilled in the art.
Other than in the operating examples, or where otherwise indicated,
all numbers expressing quantities of ingredients or reaction
conditions used herein should be understood as modified in all
instances by the term "about." The term "about" when used in
connection with percentages may mean.+-.1%.
[0026] All patents and other publications identified are expressly
incorporated herein by reference for the purpose of describing and
disclosing, for example, the methodologies described in such
publications that might be used in connection with the present
invention. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
[0027] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as those commonly understood to
one of ordinary skill in the art to which this invention pertains.
Although any known methods, devices, and materials may be used in
the practice or testing of antigenic or immunogenic non-alpha
helical PspA/PspC polypeptide embodiments, some methods, devices,
and materials in this regard are described here.
[0028] Streptococcus pneumoniae are Gram-positive, lancet-shaped
cocci (elongated cocci with a slightly pointed outer curvature).
Usually, they are seen as pairs of cocci (diplococci), but they may
also occur singly and in short chains. When cultured on blood agar,
they are alpha hemolytic. Individual cells are between 0.5
micrometers and 1.25 micrometers in diameter. They do not form
spores and they are non-motile. Like other streptococci, they lack
catalase and ferment glucose to lactic acid. Unlike other
streptococci, they do not display an M protein, they hydrolyze
inulin, and their cell wall composition is characteristic both in
terms of their peptidoglycan and their teichoic acid
compositions.
[0029] S. pneumoniae is a well known human pathogen and a major
etiologic agent for pneumonia, meningitis, otitis media as well as
sepsis, among primarily young children and older adults. Fedson
& Musher in VACCINES 2nd ED. (Plotkin & Mortimer eds., W.B.
Saunders Co., Philadelphia, Pa., 1994). A capsule composed of
polysaccharide completely envelops the pneumococcal cells. During
invasion the capsule is an essential determinant of virulence. The
capsule interferes with phagocytosis by preventing C3b opsonization
of the bacterial cells. Anti-pneumococcal vaccines are based on
formulations of various capsular (polysaccharide) antigens derived
from the highly-prevalent strains.
[0030] S. pneumoniae has been divided into ninety serotypes based
on its expression of serologically distinct carbohydrate capsules.
Henrichsen, 33 J. CLIN. MICROBIOL. 2759-62 (1995). Antibodies to a
capsular polysaccharide (PS) may provide protection against
pneumococci expressing the same capsular serotype. Currently
available pneumococcal vaccines contain a mixture of capsular PS of
multiple serotypes. For example, one pneumococcal vaccine (called
PS vaccine) contains capsular PS from twenty-three commonly found
serotypes. Robbins et al., 148 J. INFECT. DIS. 1136-59 (1983). The
most recently developed type of vaccine (called conjugate vaccine)
contains capsular PS from seven to thirteen serotypes that are
conjugated to a protein molecule. Wuorimaa & Kayhty, 56 SCAND.
J. IMMUNOL. 111-29 (2002). A seven-valent conjugate vaccine was
introduced in 2000 for clinical use in the United States, and has
reduced the incidence of invasive pneumococcal diseases in
children. Whitney, 348 N. ENGL. J. MED. 1737-46 (2003).
[0031] An alternative approach for protecting children, and also
the elderly, from pneumococcal infection employs protein antigens
that could elicit protective immune responses. Such proteins may
serve as a vaccine by themselves, may be used in conjunction with
successful polysaccharide-protein conjugates, or serve as carriers
for polysaccharide components. The pneumococcal surface protein A
(PspA) has been identified as a protein with potential for
pneumococcal vaccines. See, e.g., U.S. Pat. No. 6,592,876; No.
6,500,613; and No. 5,997,882. Indeed, the PspA protein or portions
of the protein have elicited a protective immune response following
mucosal or oral administration, particularly with an adjuvant such
as cholera toxin. See U.S. Pat. No. 6,042,838 and No.
6,232,116.
[0032] PspA is also an attractive target because it elicits mouse,
monkey, and human antibody protective in mice; is produced by all
pneumococci; interferes with complement deposition; binds
lactoferrin (inhibits apolactoferrin-killing); and is serologically
variable, yet cross-reactive.
[0033] Often, approaches for the use of PspA as a potential
protein-based pneumococcal vaccine have focused on cross-protective
epitopes found within the alpha-helical region of the PspA protein,
a region predicted to have a coiled-coil protein conformation. This
conformation, however, might possibly have potential to elicit
antibodies that cross-react with damaged human muscle proteins from
skeletal muscle and/or heart. See, e.g., Cunningham, 8 FRONT.
BIOSCI. 533-43 (2003); Cunningham, 40 MOL. IMMUNOL. 1121-27 (2004);
Krishner & Cunnimgham, 227 SCIENCE 413-15 (1985). Hence, this
has raised concerns over using PspA as an immunogen in pneumococcal
vaccines. This possibility, however, must be balanced with the
observation that adult humans may have existing antibodies to PspA,
that were raised naturally during the course of childhood
experience with Streptococcus pneumoniae. These native antibodies
have shown no correlation to rheumatic heart disease or
auto-reactive immune syndromes.
[0034] The embodiments herein provide for immunogenic
non-coiled-coil regions of PspA and PspC proteins, such as the
proline-rich regions or the choline-binding region, that are less
likely to elicit anti-myosin cross-reactive antibodies or to
stimulate any pre-existing antibodies of this type. As used herein,
PspA and PspC amino acid sequences, peptide molecules, peptides,
polypeptides, proteins, and PspA or PspC portions or fragments may
be used synonymously to refer to amino acid sequences encoded by
the PspA or PspC gene(s) that are capable of eliciting an immune
response yet lack the coiled-coil structure of myosin (although
secondary or tertiary structure of the polypeptides of the present
invention are certainly within the scope of the invention). Indeed,
the embodiments herein provide for non-alpha-helical regions of
PspA, which are also found in PspC, that are capable for eliciting
protective antibodies. These smaller portions of PspA and/or PspC
from within the alpha-helical regions may also lack the ability to
form coiled-coils (thought to perhaps elicit myosin cross-reactive
antibodies), because of their smaller size, yet retain the ability
to elicit protection. These polypeptides may be harvested from
pneumococcal cells or obtained using recombinant technologies
well-known in the art. Hence, these PspA/PspC portions may be good
candidates for protein-based pneumococcal vaccine development.
[0035] To that end, recombinant PspA/PspC polypeptides were used to
immunize mice. Mice were challenged subsequently with a lethal dose
of live S. pneumoniae. Protective responses were judged by
increased survival times. These data showed that the proline-rich
area of the choline-binding region of, as well as the non-pro-block
of the proline rich region can elicit protection. These regions
lack the coiled-coil structure implicated with a possible
anti-myosin reaction.
[0036] Within the scope of the present embodiments are derivatives
of the non-coiled-coil antigenic PspA and PspC polypeptides.
"Derivative" is intended to include modifications of the native
PspA and PspC non-alpha-coiled polypeptides that retain the either
the antigenicity or immunizing activity of the native polypeptides.
The term is intended to include, without limitation, portions,
fragments, or complexes of the protein, peptides, polypeptides, or
fusion partner proteins made by recombinant DNA or other
purification techniques whose amino acid sequences are identical or
substantially identical (i.e., differ in a manner that does not
substantially reduce the desired level of antigenicity) to that of
the protein or that of an active portion thereof, or that lack or
have different substituents (e.g., lack glycosylation or differ in
glycosylation), and conjugates of the protein or such fragments,
oligomers, polypeptides and fusion proteins and carrier proteins.
The creation and use of such polypeptides and derivatives are
well-known in the art.
[0037] It is also intended that the protein coding regions for use
in the present invention could also be provided by altering
existing PspA or PspC genes using standard molecular biological
techniques that result in variants (agonists) of the peptides
described herein. Such variants include, but are not limited to
deletions, additions and substitutions in the amino acid sequence
of the PspA/PspC peptides, and are well-known in the art.
[0038] For example, one class of substitutions is conserved amino
acid substitutions. Such substitutions are those that substitute a
given amino acid in the peptide by another amino acid of like
characteristics. Typically seen as conservative substitutions are
the replacements, one for another, among the aliphatic amino acids
Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser
and Thr, exchange of the acidic residues Asp and Glu, substitution
between the amide residues Asn and Gln, exchange of the basic
residues Lys and Arg, replacements among the aromatic residues Phe,
Tyr, and the like. Guidance concerning which amino acid changes are
likely to be phenotypically silent is found in Bowie et al., 247
SCIENCE 1306-10 (1990).
[0039] Variant or agonist peptides may be fully functional or may
lack function in one or more activities. Fully functional variants
typically contain only conservative variations or variations in
non-critical residues or in non-critical regions. Functional
variants can also contain substitution of similar amino acids that
result in no change or an insignificant change in function.
Alternatively, such substitutions may positively or negatively
affect function to some degree.
[0040] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[0041] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis. Cunningham et al., 244
SCIENCE 1081-85 (1989). The latter procedure introduces single
alanine mutations at every residue in the molecule. The resulting
mutant molecules are then tested for biological activity such as
epitope binding or in vitro ADCC activity. Sites that are critical
for ligand-receptor binding can also be determined by structural
analysis such as crystallography, nuclear magnetic resonance, or
photoaffinity labeling. Smith et al., 224 J. MOL. BIOL. 899-904
(1992); de Vos et al., 255 SCIENCE 306-12 (1992).
[0042] Moreover, polypeptides often contain amino acids other than
the twenty "naturally occurring" amino acids. Further, many amino
acids, including the terminal amino acids, may be modified by
natural processes, such as processing and other post-translational
modifications, or by chemical modification techniques well known in
the art. Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0043] Such modifications are well known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties (2nd ed., T. E.
Creighton, W. H. Freeman and Company, New York 1993). Many detailed
reviews are available on this subject, such as by Wold,
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, 1-12 (Johnson,
ed., Academic Press, New York 1983); Seifter et al., 182 METH.
ENZYMOL. 626-46 (1990); and Rattan et al., 663 ANN. N.Y. ACAD. SCI.
48-62 (1992). The secondary and tertiary structure of the peptides
of the present invention may be determined by any number of
techniques well-known in the art, or predicted by well-known
methodologies such as Chou-Fasman secondary structure analysis as
shown, for example, in FIGS. 5A-C.
[0044] Accordingly, the peptides of the present invention also
encompass derivatives or analogs in which a substituted amino acid
residue is not one encoded by the genetic code, in which a
substituent group is included (such as pegylation) as mentioned
previously.
[0045] Similarly, the additions and substitutions in the amino acid
sequence as well as variations, and modifications just described
may be equally applicable to the amino acid sequence of anti-PspA
antibodies and/or anti-PspC antibodies that may be raised against
the proteins of the present invention by methodologies well known
in the art, and are thus encompassed by the present invention.
[0046] The non-alpha-helical PspA peptides and corresponding PspC
peptides described herein may generate an immune response. The term
"immune response" refers to a cytotoxic T-cell response and/or
increased serum levels of antibodies specific to an antigen, or to
the presence of neutralizing antibodies to an antigen. The immune
response may indeed be sufficient to make the antigen of the
invention useful as a vaccine for protecting human subjects from
human pneumococcal infection. Additionally, antibodies generated by
the antigen of the invention can be extracted and used to detect a
bacterium in a body fluid sample. The term "protection" or
"protective immunity" refers herein to the ability of the serum
antibodies and/or cytotoxic T-cell response induced during
immunization to protect (partially or totally) against a disease
caused by an infectious agent, e.g., human S. pneumonaie. The use
of the immunogenic polypeptides in a vaccine is expected to provide
protective immunity to humans against severe pneumococcal infection
by inducing antibodies against pneumococci which are known to
prevent severe clinical symptoms.
[0047] In another embodiment, an immunogenic non-alpha-helical
PspA/PspC peptide is conjugated to another hatpen, thus acting as
an effective protein carrier or adjuvant for that hapten. Hapten
refers to a disease specific antigenic determinant identified by
biochemical, genetic or computational means. The haptens may be
associated with a disease condition caused by S. pneumoniae, or by
an agent such as bacteria, viruses, intracellular parasites, fungi,
and transformed (cancerous or pre-cancerous) cells.
[0048] The invention includes a method of providing an immune
response and protective immunity to a patient against
pneumococcal-mediated diseases. The method includes administering
the PspA/PspC antigen of the invention to an animal or human. The
PspA/PspC antigen of the invention is preferably administered as a
formulation comprising an effective amount of the antigen. A
variety of physiologically acceptable carriers are known in the
art, including for example, saline. Routes of administration,
amounts, and frequency of administration are known to those skilled
in the art for providing protective immunity to a recipient
subject. Routes of administration include any method which confers
protective immunity to the recipient, including, but not limited
to, inhalation, intravenous, intramuscular, intraperitoneal,
intradermal, and subcutaneous. Preferably the antigen of the
invention is provided to a human subject by subcutaneous or
intramuscular injection. A range of amounts and frequency of
administration is acceptable so long as protective immunity of the
recipient is achieved. For example, 5 .mu.g to 20 .mu.g can be
administered by intramuscular injection between one four times over
a three month period.
[0049] Hence, the novel immunogenic PspA/PspC polypeptides provided
herein may be useful in a vaccine or in pneumococcal vaccine
development. For example, the polypeptide may be incorporated into
a vaccine, either alone, as a component, or as a protein carrier
for a polysaccharide conjugate vaccine. See, e.g., U.S. Pat. No.
5,866,135; U.S. Pat. No. 5,773,007; and U.S. Pat. No.
6,936,252.
[0050] Antibodies raised against the PspA/PspC fragments of the
present invention are also encompassed herein. For example, the
monoclonal antibody designated KL67 binds to the non-pro-block
portion of PspA/PspC non-alpha helical polypeptides. The ability of
the anti-PpsA/PspC peptide antibodies to elicit cross-protection
(protection against additional strains representing different
capsular types, in which the intrinsic PspA/PspC exhibits
non-identical regions to those in the vaccine) are determined by
further immunization and challenge experiments using additional
challenge strains to verify broad cross-protection. Sera from
immunized animals such as mice and rabbits are tested for the
ability to passively protect mice to determine if the protection is
primarily through elicited antibody. Protection is further
characterized in additional mouse models (pneumonia and carriage
models), and the potential that T-cells are involved in the
protection is determined.
[0051] Additionally, the smallest portions of PspA/PspC that are
able to elicit protective antibodies may be identified in a mouse
model of pneumococcal infection. This is done by making smaller
recombinant portions, fragments, or polypeptides from the present
immunogenic regions to determine the minimal effective
epitopes.
[0052] Sera from immunized mice are also tested for the ability to
enhance complement deposition and/or to enhance lactoferrin
killing, activities that have been associated with other
anti-PspA/PspC antibodies that were found to be protective. Both
activities may be found to be elicited by one PspA/PspC fragment or
may require more than one fragment, i.e. different fragments used
in combination. These experiments may help address the assays that
need to be used as correlates of protection in vaccine
development.
[0053] Thus, for example, particular embodiments of the immunogenic
PspA/PspC polypeptides lacking the coiled-coil structure have the
amino acid sequence
DLKKAVNEPEKPAEEPENPAPAPKPAPAPQPEKPAPAPAPKPEKSADQQAEEDYARR
SEEEYNRLTQQQPPKAEKPAPAPVPKPEQPAPAPKTGWGQENGMW [SEQ ID NO: 1];
DLKKAVNEPETPAPAPAPAPAPAPTPEAPAPAPAPAPKPAPAPKPAPAPKPAPAPKPA
PAPKPAPAPKPAPAPAPAPKPEKPAEKPAPAPKPETPKTGWKQENGMW [SEQ ID NO:2];
MAKKAELEKTPEKPAEEPENPAPAPQPEKSADQQAEEDYARRSEEEYNRLTQQQPPK A [SEQ ID
NO: 3]; or EKSADQQAEEDYARRSEEEYNRLTQQQ [SEQ ID NO: 4].
[0054] The sequence EKSADQQAEEDYARRSEEEYNRLTQQQ [SEQ ID NO: 4] is
also referred to as the non-pro-block (NPB) having no proline amino
acid residues in an otherwise proline-rich area of the non coiled
region of PspA/PspC. This peptide is also identified by binding
with the monoclonal antibody designated KL67, which also binds to
DLKKAVNEPEKPAEEPENPAPAPKPAPAPQPEKPAPAPAPKPEKSADQQAEEDYARR
SEEEYNRLTQQQPPKAEKPAPAPVPKPEQPAPAPKTGWGQENGMW [SEQ ID NO: 1] and
MAKKAELEKTPEKPAEEPENPAPAPQPEKSADQQAEEDYARRSEEEYNRLTQQQPPK A [SEQ ID
NO: 3]. As such, EKSADQQAEEDYARRSEEEYNRLTQQQ [SEQ ID NO: 4] may
also be considered an example portion of an immunogenic PspA/PspC
peptide as disclosed herein.
[0055] Note also that the sequence,
MAKKAELEKTPEKPAEEPENPAPAPQPEKSADQQAEEDYARRSEEEYNRLTQQQPPK A [SEQ ID
NO: 3], is an example of a semi-artificial variant or derivative of
a native PspA/PspC sequence, having the SM-I amino acid sequence
AAKKAELEKT [SEQ ID NO: 5] added to it the native sequence. See
Example 1, below.
[0056] Another example variant of the NPB has the amino acid
sequence MEKSADQQAEEDYARRSEEEYNRLTQQQ [SEQ ID NO: 6], which has a
methionine on the amino-terminus as it may be expressed in an
expression vector.
[0057] A particular embodiment provides for an immunogenic
recombinant peptide comprising the amino acid sequence
MEKSADQQAEEDYARRSEEEYNRLTQQQ [SEQ ID NO: 6]. A variant of this
sequence, for example, with a conservative amino acid substitution
in which V is substituted for A at position 5, is
TABLE-US-00001 MEKSVDQQAEEDYARRSEEEYNRLTQQQ [SEQ ID NO: 7]
[0058] Prior to the embodiments described herein, it was known that
many pspA and pspC genes expressed similar structures (i.e. PAP . .
. repetitive proline rich stretches--with or without the
EKSADQQAEEDYARRSEEEYNRLTQQQ [SEQ ID NO: 4]. The amino acids at
either end of these peptides mark the boundaries of the proline
rich region. Amino-terminal to the region is DLKKAVNE . . . [SEQ ID
NO: 8], carboxy-terminal to it is (K/G)TGW(K/G)QENGMW [SEQ ID NO:
9]. Peptides containing the NPB are immunogenic, suggesting that
the NPB (e.g., SEQ ID NO: 4) may be an important epitope. This
sequence of amino acids is also accessible to antibody binding from
the surface of the bacterium, see Example 3, FIG. 9, depicting FACS
data. Surface binding is likely to be important for protection, and
a factor in future vaccine development.
[0059] PCR-based evidence indicates that about 77% of the pspC
genes about 50% of pspA genes in isolates from Africa, Asia, and
South America contain the NPB sequence (e.g.,
EKSADQQAEEDYARRSEEEYNRLTQQQ [SEQ ID NO: 4]) or a close homolog of
this peptide. Hence these homologs represent alternative
embodiments. Additionally, the NPB constitutes a common epitope
present among most of the bacterial strains for which a vaccine is
needed.
[0060] Nucleic acids that encode one or more of the
non-alpha-helical PspA/PspC peptides, polynucleotides sufficient
for use as hybridization probes, PCR primers or sequencing primers
for identifying, analyzing, mutating or amplifying a polynucleotide
encoding a polypeptide, anti-sense nucleic acids for inhibiting
expression of a polynucleotide, and complementary sequences of the
foregoing are also provided. The nucleic acids can be any length
and/or can comprise one or more additional sequences, for example,
regulatory sequences, and/or be part of a larger nucleic acid, for
example, a vector. The nucleic acids can be single-stranded or
double-stranded and can comprise RNA and/or DNA nucleotides, and
artificial variants thereof (e.g., peptide nucleic acids). For
example, nucleic acids (DNAs) encompassed herein include those
encoding the PAC001 peptide (see Example 1, below), having the
sequence:
TABLE-US-00002 [SEQ ID NO: 10]
GACCTTAAGAAAGCAGTTAATGAGCCAGAAAAACCAGCTGAAGAGCCTGA
GAATCCAGCTCCTGCACCAAAACCAGCGCCGGCTCCTCAACCAGAAAAAC
CAGCTCCAGCTCCTGCACCAAAACCAGAGAAGTCAGCAGATCAACAAGCT
GAAGAAGACTATGCTCGTAGATCAGAAGAAGAATATAACCGCTTGACTCA
ACAGCAACCGCCAAAAGCAGAAAAACCAGCTCCAGCTCCTGTACCAAAAC
CAGAGCAACCAGCTCCCGCACCAAAAACGGGCTGGGGACAAGAAAACGGT ATGTGG
[0061] An example DNA encoding the PAC003 peptide has the
sequence:
TABLE-US-00003 [SEQ ID NO: 11]
GACCTTAAGAAAGCAGTTAATGAGCCAGAAACTCCAGCTCCGGCTCCAGC
CCCAGCTCCAGCTCCAGCTCCAACTCCAGAAGCCCCAGCTCCAGCTCCAG
CTCCGGCTCCTAAACCAGCTCCGGCTCCTAAACCAGCTCCGGCTCCTAAA
CCAGCTCCGGCTCCTAAACCAGCTCCGGCTCCTAAACCAGCTCCGGCTCC
TAAACCAGCTCCAGCTCCAGCTCCGGCTCCTAAACCAGAAAAGCCAGCAG
AAAAACCAGCTCCAGCTCCTAAACCAGAAACTCCAAAAACAGGCTGGAAA
CAAGAAAACGGTATGTGG
[0062] An example DNA encoding the MB001 peptide has the
sequence:
TABLE-US-00004 [SEQ ID NO: 12]
ATGGCTAAAAAAGCTGAATTAGAAAAAACTCCAGAAAAACCAGCTGAAGA
GCCTGAGAATCCAGCTCCAGCACCACAACCAGAGAAGTCAGCAGATCAAC
AAGCTGAAGAAGACTATGCTCGTAGATCAGAAGAAGAATATAATCGCTTG
ACCCAACAGCAACCGCCAAAAGCA
[0063] An example DNA encoding a NPB (from MB001) has the
sequence:
TABLE-US-00005 [SEQ ID NO: 13]
GAGAAGTCAGCAGATCAACAAGCTGAAGAAGACTATGCTCGTAGATCAGA
AGAAGAATATAATCGCTTGACCCAACAGCAACCG
[0064] Another example DNA encoding a NPB (in PAC001) has the
sequence:
TABLE-US-00006 [SEQ ID NO: 14]
GAGAAGTCAGCAGATCAACAAGCTGAAGAAGACTATGCTCGTAGAT
CAGAAGAAGAATATAACCGCTTGACTCAACAGCAACCG
[0065] The invention will now be described further by non-limiting
examples.
EXAMPLES
Example 1
Recombinant PspA Polypeptides
[0066] Recombinant plasmids producing some of the relevant PspA
polypeptides were generated and used to provide purified rPspA
proteins. The proteins made are indicated in FIG. 1, and detailed
amino acid sequences are given in FIGS. 2A-E and FIGS. 3A-D.
Briefly, the expression vectors include a thioredoxin gene (trx)
from mouse. The gene fragment encoding the peptide of interest is
cloned in such a way that it makes a protein fusion with the
thioredoxin. In FIGS. 2A-E, the non-underlined amino acids came
from the vector. The underlined amino acids came from the cloned
portion. The PET32A and PET32B proteins are referred to as "fusion
partner."
Construction of the Clone pPAC001 and Purification of
PspA/AC94.sub.AAPro-rich.
[0067] The protein is called PAC001 (or PspA/AC94Pro). This
recombinant protein contains the proline-rich region of the pspA
gene from strain AC94. This is a proline-rich region that has a NPB
in the center of two proline-rich stretches of amino acids. The NPB
has the amino acid sequence DQQAEEDYARRSEEEYNRLTQQQ [SEQ ID NO:
15]. Robinson et al., 2001.
[0068] Construction of vector. An internal gene fragment of the
pspA.AC94 gene (see Robinson et al., Clones of Streptococcus
pneumoniae isolated from nasophayngeal carriage and invasive
disease in young children in Tennessee, 183 J. INFECT. DIS. (2001))
encoding PspA/AC94pro was amplified by polymerase chain reaction
from the Streptococcus pneumoniae strain AC94 using the
oligonucleotides 5'GGGAGCCATGGCTGACCTTAAGAAAGCAGTTAATGAGCCA3' [SEQ
ID NO: 16] (pspA27-NcoI) and
5'CCGTCGACACCACATACCGTTTTCTTGTTTCCAGCC3' [SEQ ID NO: 17]
(pspA22-Sal1) (restriction endonuclease (RE) recognition sites
underlined). Reactions were carried out for 30 cycles in a total
volume of 50 ml in a cocktail containing 3.0 mM MgCl2, 125 mM
dNTPs, 50 picomole of each primer, and 2.5 units of Taq DNA
Polymerase. The cycle was 94.degree. C., 1 min.; 55.degree. C., 1
min.; 72.degree. C., 5 min. This amplified gene fragment was
digested with NcoI and SalI, and the .about.300 bp pspA gene
fragment was then incorporated between NcoI and SalI sites of a
vector (pET32a, Novagen, Inc.) with a strong T7 promoter and
translation signals. DNA sequence confirmed that the recombinant
plasmid pPAC001 contained the expected 315 kbp pspA gene fragment
inserted after the trx gene and the His-tag site in vector
pET32a.
[0069] The plasmid pPAC001 was transformed into the E. coli strain
BL21 STAR (DE3) for protein production. This strain contains a
chromosomal copy of the T7 promoter under control of the inducible
UV5 promoter. Upon induction with IPTG, a recombinant protein that
contains 283 amino acids, 105 of which derive from the proline-rich
region of PspA protein, is expressed. The rPAC001 is a protein
fusion between thioredoxin (from vector) and the proline-rich
region. The sequence of the complete recombinant protein is given
below (italics proline insert only). The six histidine residues
present in the middle of the recombinant protein are used to
simplify its purification by nickel chromatography. It can also be
cleaved with enterokinase to allow its release from the trx fusion
partner. The protein was made in this fashion to ensure
immunogenicity because of the small size of the PspA-specific
portion. The rPET32a protein (mostly thioredoxin) is used as a
negative control immunogen in immunization protocols.
[0070] The protein sequence of the insert is:
TABLE-US-00007
MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVA [SEQ ID
NO: 18] KLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSG
HMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADLKKAVNE
PEKPAEEPENPAPAPKPAPAPQPEKPAPAPAPKPEKSADQQAEEDYARRSEEEYNRLTQQ
QPPKAEKPAPAPVPKPEQPAPAPKTGWGQENGMWCRQACGRTRAPPPPPLRSGC
[0071] The thioredoxin gene and the His tag, plus some sequences
the N-terminal end and the C-terminal end are from the pET32a
vector. The total amino acid composition is included herein, the
105 amino acids in italics, above, are the native PspA amino acids.
Note that about one-quarter of these amino acids are proline.
[0072] The DNA sequence of the PCR product before cutting with NcoI
and SalI is:
TABLE-US-00008 [SEQ ID NO: 19]
xxxCCATGGCTGACCTTAAGAAAGCAGTTAATGAGCCAGAAAAACCAGCT
GAAGAGCCTGAGAATCCAGCTCCTGCACCAAAACCAGCGCCGGCTCCTCA
ACCAGAAAAACCAGCTCCAGCTCCTGCACCAAAACCAGAGAAGTCAGCAG
ATCAACAAGCTGAAGAAGACTATGCTCGTAGATCAGAAGAAGAATATAAC
CGCTTGACTCAACAGCAACCGCCAAAAGCAGAAAAACCAGCTCCAGCTCC
TGTACCAAAACCAGAGCAACCAGCTCCCGCACCAAAAACGGGCTGGGGAC
AAGAAAACGGTATGTGGTGTCGACxxx
[0073] This includes the NcoI sites and SalI sites that were on the
primers. (RE sites underlined in above sequence).
[0074] An example nucleic acid (DNA) encoding the PAC001
polypeptide has the sequence:
TABLE-US-00009 [SEQ ID NO: 10]
GACCTTAAGAAAGCAGTTAATGAGCCAGAAAAACCAGCTGAAGAGCCTGA
GAATCCAGCTCCTGCACCAAAACCAGCGCCGGCTCCTCAACCAGAAAAAC
CAGCTCCAGCTCCTGCACCAAAACCAGAGAAGTCAGCAGATCAACAAGCT
GAAGAAGACTATGCTCGTAGATCAGAAGAAGAATATAACCGCTTGACTCA
ACAGCAACCGCCAAAAGCAGAAAAACCAGCTCCAGCTCCTGTACCAAAAC
CAGAGCAACCAGCTCCCGCACCAAAAACGGGCTGGGGACAAGAAAACGGT ATGTGG
[0075] An example nucleic acid encoding the NPB within the PAC001
polypeptide has the sequence:
TABLE-US-00010 [SEQ ID NO: 14]
GAGAAGTCAGCAGATCAACAAGCTGAAGAAGACTATGCTCGTAGATCAGA
AGAAGAATATAACCGCTTGACTCAACAGCAACCG
TABLE-US-00011 Calculated Molecular Weight = 30531.77 Estimated pI
= 5.56 Amino Acid Composition: No. Percent Non-polar: A 36 12.72 V
8 2.83 L 18 6.36 I 9 3.18 P 37 13.07 M 7 2.47 F 5 1.77 W 4 1.41
Polar: G 21 7.42 S 12 4.24 T 11 3.89 C 5 1.77 Y 4 1.41 N 8 2.83 Q
13 4.59 Acidic: D 20 7.07 E 22 7.77 Basic: K 24 8.48 R 10 3.53 H 9
3.18
[0076] An ELISA method is used to determine antibody titers to PspA
proline-rich portion. Initial tests of this antigen in ELISA, and
antibody response directed at the fusion partner protein (pET32a),
are measured. An inhibition ELISA in which most of the
pET32a-specific response is inhibited is used in order to get data
of PspA-specific response. Based upon total protein concentrations
determined by the Biorad protein assay, Nunc MaxiSorp plates (Nalge
Nunc Int'l, Denmark) are coated with 3 .mu.g per ml of the
recombinant protein in phosphate buffered saline overnight. Control
wells are coated with a similar amount of pET32a alone. A 1:1000
dilution of a mouse is used in ELISA. Non-specific reactivity is
inhibited by adding 3-10 ug/mL of pET32a protein in with the sera
diluent. This is constant across the sera dilution.
[0077] Additional references relating to PspA clades and families
include Hollingshead et al., 68 INFECT. IMMUN. 5889-900 (2000);
Briles et al., 182 J. INFECT. DIS. 1694-1791 (2000); and Briles et
al., 18 VACCINE 1707-11 (2000). Further information relating to the
pspC gene is available, for example, see Brooks-Walter et al., 67
INFECT. IMMUN. 6533-6542 (1999).
Construction of the Clone pPAC003 and Purification of
PspA/BG9739.sub.AAPro-rich
[0078] The protein is called PAC003 (or PspA/BG9739.sub.Pro). This
recombinant protein contains the proline-rich region of the pspA
gene from strain BG9739. This is a proline-rich regions that does
not have a NPB in the center of two proline-rich stretches of amino
acids. The NPB was that of SEQ ID NO: 15. The NPB is an immunogenic
section of PspA, hence the inventors differentiate whether
protective antibodies react with proline-rich epitopes or perhaps
others in this region using this construct.
[0079] In constructing a vector, an internal gene fragment of the
pspA.BG9739 gene encoding PspA/BG9739Pro, was amplified by
polymerase chain reaction from the S. pneumoniae strain BG9739
using the oligonucleotides of SEQ ID NO: 16 and SEQ ID NO: 17.
Reactions were carried out as with the construction of pPAC001. DNA
sequence confirmed that the recombinant plasmid pPAC003 contained
the expected 334 kbp pspA gene fragment inserted after the trx gene
and the His-tag site in vector pET32a.
[0080] The plasmid pPAC003 was transformed into the E. coli strain
BL21 AI (DE3) for protein production. This strain contains a
chromosomal copy of the T7 promoter under control of the inducible
araBAD promoter. Upon induction with arabinose and IPTG, a
recombinant protein that contains 288 amino acids, 110 of which
derive from the proline-rich region of PspA_BG9739 protein, is
expressed. The rPAC003 is a protein fusion between thioredoxin
(from vector) and the proline-rich region. The sequence of the
complete recombinant protein is given below (italics proline insert
only). The six histidine residues present in the middle of the
recombinant protein are used to simplify its purification by nickel
chromatography. It can also be cleaved with enterokinase to allow
its release from the trx fusion partner. The protein was made in
this fashion to ensure immunogenicity because of the small size of
the PspA-specific portion. The rPET32a protein (mostly thioredoxin)
is used as a negative control immunogen in immunization
protocols.
[0081] The protein sequence of the insert is:
TABLE-US-00012
MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVA [SEQ ID
NO: 20] KLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSG
HMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADLKKAVNE
PETPAPAPAPAPAPAPTPEAPAPAPAPAPKPAPAPKPAPAPKPAPAPKPAPAPKPAPAPKP
APAPAPAPKPEKPAEKPAPAPKPETPKTGWKQENGMWCRQACGRTRAPPPPPLRSG
[0082] The thioredoxin gene and the His tag, plus a bit more at the
N-terminal end and a bit at the C-terminal end are coming from the
pET32a vector. The total amino acid composition follows below. Of
the amino acid sequence above, 109 (in italics, above) are the
native PspA amino acids. About one-third of that is proline. The
DNA sequence of the PCR product before cutting with NcoI and SalI
is:
TABLE-US-00013 [SEQ ID NO: 21]
xxxCATGGCTGACCTTAAGAAAGCAGTTAATGAGCCAGAAACTCCAGCTC
CGGCTCCAGCCCCAGCTCCAGCTCCAGCTCCAACTCCAGAAGCCCCAGCT
CCAGCTCCAGCTCCGGCTCCTAAACCAGCTCCGGCTCCTAAACCAGCTCC
GGCTCCTAAACCAGCTCCGGCTCCTAAACCAGCTCCGGCTCCTAAACCAG
CTCCGGCTCCTAAACCAGCTCCAGCTCCAGCTCCGGCTCCTAAACCAGAA
AAGCCAGCAGAAAAACCAGCTCCAGCTCCTAAACCAGAAACTCCAAAAAC
AGGCTGGAAACAAGAAAACGGTATGTGGTGTCGACxxx
[0083] This includes the NcoI sites and SalI sites that were on the
primers (RE sites underlined in above sequence).
TABLE-US-00014 A relevant nucleic acid (DNA) encoding the PAC003
polypeptide has the sequence: [SEQ ID NO: 11]
GACCTTAAGAAAGCAGTTAATGAGCCAGAAACTCCAGCTCCGGCTCCAGC
CCCAGCTCCAGCTCCAGCTCCAACTCCAGAAGCCCCAGCTCCAGCTCCAG
CTCCGGCTCCTAAACCAGCTCCGGCTCCTAAACCAGCTCCGGCTCCTAAA
CCAGCTCCGGCTCCTAAACCAGCTCCGGCTCCTAAACCAGCTCCGGCTCC
TAAACCAGCTCCAGCTCCAGCTCCGGCTCCTAAACCAGAAAAGCCAGCAG
AAAAACCAGCTCCAGCTCCTAAACCAGAAACTCCAAAAACAGGCTGGAAA
CAAGAAAACGGTATGTGG
TABLE-US-00015 Calculated Molecular Weight = 29795.33 Estimated pI
= 8.48 Amino Acid Composition: No. Percent Non-polar: A 48 16.78 V
7 2.45 L 17 5.94 I 9 3.15 P 53 18.53 M 7 2.45 F 5 1.75 W 4 1.40
Polar: G 20 6.99 S 10 3.50 T 13 4.55 C 4 1.40 Y 2 0.70 N 6 2.10 Q 6
2.10 Acidic: D 18 6.29 E 14 4.90 Basic: K 27 9.44 R 7 2.45 H 9
3.15
[0084] This construct may be used to test whether this portion of
PspA can elicit protective antibodies in animal model systems and
in humans. Additionally, this is useful to test how broad the
protection is (e.g., can it protect against strains with different
PspA/PspC proteins). It may differentiate between proline-rich
epitopes and others that may fall in this region of the PspA
protein.
Construction of the Clone pMB001 and Purification of
PspA.sub.SM-1.sub.--.sub.Pro-rich.
[0085] The protein is called MB001 (or PspASM-1_non-pro-block).
This recombinant protein contains both the non-pro-block
(Brooks-Walter et al., 1999) present in some of the proline-rich
regions of the pspA gene along with the peptide SM-1 (Shaper et
al., 72 Infect. Immun. 5031-40 (2004)), and some small remnants
from the proline-rich segment. The NPB has the amino acid sequence
of SEQ ID NO: 15 and the sequence called SM-1 is
TABLE-US-00016 AAKKAELEKT. [SEQ ID NO: 7]
[0086] These two regions were of interest because of the following
observations: (1) Non-pro block: in immunizations with various
rPspC proteins, many of which contained this block, the inventors
were able to immunize and protect against challenge strains which
lacked PspC but had a PspA with a very similar non-pro-block. Using
UAB055 and UAB103 (two proteins that differ only by the
non-pro-block) in ELISA, it was found that immunization with rPspC
elicited antibodies that reacted with the non-pro-block in UAB103.
Some screens of the cross-reactivity among various BC100-type
antiseras suggested that the non-pro block was more immunogenic
than the specific proline-rich segments around it. The block was
present in 15/24 PspA genes sequenced and in 10/12 PspC genes
sequenced; (2) SM-1 peptide: it has been found that apo-lactoferrin
can kill pneumococci, but this killing could be inhibited by the
SM-1 peptide (but not by other peptides). This peptide may (or may
not) represent the region where PspA binds to lactoferrin. This
small region is the best conserved segment of the alignment between
four clades of PspA:
TABLE-US-00017 [SEQ ID NO: 22] Clade 1 I . . . AKKAELEKA . . . EAD
[SEQ ID NO: 23] Clade 2 A . . . AKKAELEKT . . . EAD [SEQ ID NO: 24]
Clade 3 A . . . TKKAELEKT . . . QKE [SEQ ID NO: 25] Clade 4 A . . .
TKKAELEKT . . . QKE
(3) Region related to proline-rich segments in pMB001: this
construct also has the following small segment PEKPAEEPENPAPAPQPEKS
[SEQ ID NO: 26], which came from a Brazilian strain. Each
proline-rich segment may differ between strains, but PAPAP [SEQ ID
NO: 27] and PEKPA [SEQ ID NO: 28] are often seen in reiterative
patterns. Another cloned protein, PAC003, addresses the
antigenicity of this region.
[0087] In constructing a vector, an internal gene fragment was cut
using EcoRI from a previous clone (see below). pET32b was cut with
EcoRI and phosphatased. The two were ligated together and
transformants were screened for the proper orientation. The screen
was done using primer 5'GGTACCTGCTTTTGGCGGTTGCTG3' [SEQ ID NO: 29]
CL1pro1. Together with T7prom primer, this gives about a 783 bp PCR
fragment in the proper orientation. In this orientation it makes
the fusion protein presented below.
[0088] Sequences relevant to pMB001, PspA specific portion
TABLE-US-00018 Xho 1 [SEQ ID NO: 30] 5' CTC GAG ATG GCT AAA AAA GCT
GAA TTA GAA AAA ACT CCA GAA AAA CCA GCT GAA GAG CCT GAG AAT CCA GCT
TCA GCA CCA CAA CCA CAA CAA GCT GAA GAA GAC TAT GCT CGT AGA TCA GAA
GAA GAA TAT AAT CGC TTG ACC CAA GCA GGA AAA ACC AGC 3'
[0089] Because this fragment was cut by EcoRI from pGem-T, this
piece was inserted into the EcoR1-cut pET32b to make pMB001.
Orientation was checked with primers CL1pro1--together with T7prom
(yielding about a 783 bp PCR fragment). In this orientation--it
makes the fusion protein below:
TABLE-US-00019 [SEQ ID NO: 31] EcoR1 Xho1 5' GAATTCACTAGTGATT CTC
GAG ATG GCT AAA AAA GCT GAA TTA GAA L E M A K K AAA ACT CCA GAA AAA
CCA GCT GAA GAG CCT GAG AAT CCA GCT CCA GCA CCA CAA CCA GAG AAG TCA
GCA GAT CAA CAA GCT GAA GAA GAC TAT GCT CGT AGA TCA GAA GAA GAA TAT
AAT CGC TTG ACC CAA CAG CAA CCG CCA AAA GCA GGT ACC AATCGAATTC
EcoR1 Kpn 1
[0090] Protein produced in pMB001:
TABLE-US-00020 [SEQ ID NO: 32]
MSDKHHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQ
GKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLK
EFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDL
GTDDDDKAMAISDPNSLVILEMAKKAELEKTPEKPAEEPENPAPAPQPEK
SADQQAEEDYARRSEEEYNRLTQQQPPKAGTNRIRAPSTSLRPHSSTTTT
TTEIRLLTKPERKLSWLLPPLSNN
[0091] Of the amino acids presented above, the italics signify the
trx protein; underline designates the
His-tag_thrombin_S-Tag_enterokinase region; plain text shows the
portion carried along from pGEM-T; and bold signifies the
PspA-related portion, containing the SM1 peptide and part of a
proline region.
[0092] In this instance, the DNA sequence confirmed that the
recombinant plasmid pMB001 contains the 162 bp from pspA genes (two
discontinuous fragments) which are inserted after the trx gene and
the His-tag site in vector pET32b. These lead to 54 AA of PspA in
the final recombinant protein.
[0093] pMB001 was transformed into the E. coli strain BL21 STAR
(DE3) for protein production. This strain contains a chromosomal
copy of the T7 promoter under control of the inducible UV5
promoter. Upon induction with IPTG, a recombinant protein that
contains 275 amino acids, 54 of which derive from the PspA protein,
is expressed. The rPAC001 is a protein fusion between thioredoxin
(from vector) and the proline-rich region. The sequence of the
complete recombinant protein is given below (italics proline insert
only). The six histidine residues present in the middle of the
recombinant protein are used to simplify its purification by nickel
chromatography. It can also be cleaved with enterokinase to allow
its release from the trx fusion partner. The protein was made in
this fashion to ensure immunogenicity because of the small size of
the PspA-specific portion. The rPET32a protein (mostly thioredoxin)
is used as a negative control immunogen in immunization
protocols.
[0094] An example nucleic acid (DNA) encoding the MB001 polypeptide
has the sequence:
TABLE-US-00021 [SEQ ID NO: 12]
ATGGCTAAAAAAGCTGAATTAGAAAAAACTCCAGAAAAACCAGCTGAAGA
GCCTGAGAATCCAGCTCCAGCACCACAACCAGAGAAGTCAGCAGATCAAC
AAGCTGAAGAAGACTATGCTCGTAGATCAGAAGAAGAATATAATCGCTTG
ACCCAACAGCAACCGCCAAAAGCA
[0095] Additionally, an example nucleic acid (DNA) encoding the NPB
peptide has the sequence:
TABLE-US-00022 [SEQ ID NO: 13]
GAGAAGTCAGCAGATCAACAAGCTGAAGAAGACTATGCTCGTAGATCAGA
AGAAGAATATAATCGCTTGACCCAACAGCAACCG
TABLE-US-00023 Calculated Molecular Weight = 30141.38 Estimated pI
= 5.40 Amino Acid Composition: No. Percent Non-polar: A 27 9.82 V 7
2.55 L 26 9.45 I 13 4.73 P 22 8.00 M 7 2.55 F 5 1.82 W 3 1.09
Polar: G 17 6.18 S 19 6.91 T 19 6.91 C 2 0.73 Y 4 1.45 N 10 3.64 Q
10 3.64 Acidic: D 20 7.27 E 22 8.00 Basic: K 21 7.64 R 11 4.00 H 10
3.64
[0096] This construct is used to test whether adding a portion of
PspA (SM-1, in particular) may elicit superior protective
antibodies in animal model systems and in humans as compared to
immunogens that contain the proline-rich subsections alone (such as
PAC001 or PAC003). The breadth of protection (e.g., can it protect
against strains with different PspAs) may also be determined.
Construction of the Clone pCD1_NPB and Purification of GST_NPB
Peptide
[0097] The expressed from this construct is called GST_NPB (or,
alternatively, NPB Protein). This recombinant protein contains the
NPB that is embedded in the proline-rich region of some PspA and
some PspC genes. This particular construct was cloned from pPAC001
(strain AC94). An amino acid sequence referred to as the NPB is,
for example, MEKSADQQAEEDYARRSEEEYNRLTQQQ[SEQ ID NO: 6]. It appears
that this NPB is a highly immunogenic section in this polypeptide,
and may raise antibodies that are protective and that bind the
surface of encapsulated cells.
[0098] The vector was constructed as follows: An internal gene
fragment of the PspA/AC94pro gene encoding PspA/AC94pro, was
amplified by polymerase chain reaction from the pPAC001 plasmid
using the oligonucleotides 5'-GACGACGACAAGATGGAG AAGTCAGCAGATCAA-3'
[SEQ ID NO: 33] (non-pro-L5) and 5'-GAGGAGAAGCCC
GGTTTACCGTTGCTGTTGAGTCAAGCG3' [SEQ ID NO: 34]
(non-pro-R3-corrected). The corrected sequence added the TTA to
provide a stop codon. PCR reactions were carried out for 30 cycles
in a total volume of 50 ml in a cocktail containing 3.0 mM
MgCl.sub.2, 125 mM dNTPs, 0.4 umol final concentration of each
primer, and 0.4 units of KOD DNA Polymerase. The amplicon from this
reaction was treated with T4 DNA polymerase as per pET41
man'facturer's protocol. The cycle was 94.degree. C., 1 min.;
55.degree. C., 1 min; 72.degree. C., 5 minutes. This amplified gene
fragment was incorporated by ligation independent cloning at the
EK/LIC site of a vector (pET41 EK/LIC, Novagen, Inc.) with a strong
T7 promoter and translation signals. DNA sequence confirmed that
the recombinant plasmid pCD--NPB containing the expected .about.117
bp pspA gene fragment inserted after the GST gene and the His-tag
site in vector pET41 EK/LIC.
[0099] The plasmid pCD_NPB was transformed into the E. coli strain
BL21 STAR (DE3) for protein production. This strain contains a
chromosomal copy of the T7 promoter under control of the inducible
UV5 promoter. Upon induction with IPTG, a recombinant protein that
contains 307 amino acids, 28 of which derive from the proline-rich
region of PspA protein, is expressed. The rCD_NPB is a protein
fusion between glutathione-S-transferase (GST-Tag) (from vector)
and the non-pro-block of the proline-rich region. The sequence of
the complete recombinant protein is given below (underlined). The
six histidine residues present in the middle of the recombinant
protein are used to simplify its purification by nickel
chromatography. It can also be cleaved with thrombin or
enterokinase to allow its release from the GST fusion partner. The
protein was made in this fashion to ensure immunogenicity because
of the small size of the PspA-specific portion. The rPET41 protein
(mostly GST) is used as a negative control immunogen in
immunization protocols.
[0100] The protein sequence of the insert is:
TABLE-US-00024 [SEQ ID NO: 35]
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGL
EFPNLPYYIDGDVKLTOSMAIIRYIADKHNMLGGCPKERAEISMLEGAVL
DIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTH
PDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIA
WPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSAGLVPRGSTAIGMKET
AAAKFEROHMDSPDLGTGGGSGDDDDKMEKSADQQAEEDYARRSEEEYNR LTQQQP
[0101] The GST gene, the His tag, the thrombin site, the S-Tag,
plus a bit more at the N-terminal end are coming from the pET41
Ek/LIC vector. The total amino acid composition is presented.
Twenty-eight of the amino acids (plain text, not underlined) are
the native PspA amino acids from the non-pro-block.
[0102] The DNA sequence of the PCR product with LIC ends before
using T4 DNA polymerase to exonucleolytically digest the ends
was:
TABLE-US-00025 [SEQ ID NO: 36]
GACGACGACAAGATGGAGAAGTCAGCAGATCAACAAGCTGAAGAAGACTA
TGCTCGTAGATCAGAAGAAGAATATAATCGCTTGACCCAACAGCAACCGT
AAACCGGGCTTCTCCTC
[0103] This includes the LIC sites that were on the primers (RE
sites underlined in above sequence).
TABLE-US-00026 Calculated Molecular Weight = 34983.93 Estimated pI
= 5.58 Amino Acid Composition: No. Percent Non-polar: A 18 5.88 V
10 3.27 L 30 9.80 I 14 4.58 P 16 5.23 M 12 3.92 F 10 3.27 W 4 1.31
Polar: G 25 8.17 S 18 5.88 T 11 3.59 C 4 1.31 Y 16 5.23 N 5 1.63 Q
11 3.59 Acidic: D 26 8.50 E 24 7.84 Basic: K 25 8.17 R 14 4.58 H 13
4.25
[0104] The similarity of the non-pro-block in multiple pspA and
pspC genes is indicated in FIGS. 12A-B. These polypeptides, having
homologous sequences, also fall within the scope of the immunogenic
non-alpha-coiled PspA/PspC polypeptide embodiments described
herein. The variations in these amino acid sequences may also be
considered derivatives, variants, or portions of the immunogenic
polypeptides described herein.
Construction of the Clone pCD42_NPB and Purification of GST_NPB
Peptide
[0105] The protein expressed from this clone was designated GST_NPB
(or NPB Protein). This recombinant protein contains the NPB that is
embedded in the proline-rich region of some pspA and some pspC
genes. This particular one was cloned from pPAC001 (strain AC94).
The amino acid sequence of the NPB in this construct is
MEKSADQQAEEDYARRSEEEYNRLTQQQ [SEQ ID NO: 6]. The NPB is
immunogenic, antibodies to it are protective and also bind from the
surface of encapsulated cells.
[0106] Construction of vector. An internal gene fragment of the
PspA/AC94Pro gene1 encoding PspA/AC94Pro, was amplified by
polymerase chain reaction from the pPAC001 plasmid using the
oligonucleotides 5' GACGACGACAAGATGGAGAAGTCAGCAGAT CAA3' [SEQ ID
NO: 37] (non-pro-L5) and 5'GAGGAGAAGCCCGGTTTACCGTTGCTG
TTGAGTCAAGCG3' [SEQ ID NO: 38] (non-pro-R3-corrected). The
correction added TTA to provide a stop codon. PCR reactions were
carried out for 30 cycles in a total volume of 50 ml in a cocktail
containing 3.0 mM MgCl2, 125 mM dNTPs, 0.4 umol final concentration
of each primer, and 0.4 units of KOD DNA Polymerase. The amplicon
from this reaction was treated with T4 DNA polymerase as per pET41
manufacturer's protocol. The cycle was 94.degree. C., 1 min.;
55.degree. C., 1 min; 72.degree. C., 5 min. This amplified gene
fragment was incorporated by ligation independent cloning at the
EK/LIC site of a vector (pET41 EK/LIC, Novagen, Inc.) with a strong
T7 promoter and translation signals. DNA sequencing confirmed that
the recombinant plasmid pCD--NPB containing the expected .about.117
bp pspA gene fragment inserted after the GST gene and the His-tag
site in vector pET41 EK/LIC.
[0107] The plasmid pCD42_NPB was transformed into the E. coli
strain BL21 STAR (DE3) for protein production. This strain contains
a chromosomal copy of the T7 promoter under control of the
inducible UV5 promoter. Upon induction with IPTG, a recombinant
protein that contains 307 amino acids, 28 of which derive from the
proline-rich region of PspA protein, is expressed. The rCD_NPB is a
protein fusion between glutathione-S-transferase (GST-Tag) (from
vector) and the non-pro-block of the proline-rich region. The
sequence of the complete recombinant protein is given below
(italics insert only). The six histidine residues present in the
middle of the recombinant protein are used to simplify its
purification by nickel chromatography. It can also be cleaved with
thrombin or enterokinase to allow its release from the GST fusion
partner. The protein was made in this fashion to ensure
immunogenicity because of the small size of the PspA-specific
portion. The rPET41 protein (mostly GST) is used as a negative
control immunogen in immunization protocols.
[0108] The protein sequence of the insert is:
TABLE-US-00027 [SEQ ID NO: 39]
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGL
EFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVL
DIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTH
PDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIA
WPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSAGLVPRGSTAIGMKET
AAAKFERQHMDSPDLGTGGGSGDDDDKMEKSADQQAEEDYARRSEEEYNR LTQQQP
[0109] The GST gene, the His tag, the thrombin site, the S-Tag,
plus a bit more at the N-terminal end come from the pET41 Ek/LIC
vector. Analysis of the total amino acid composition is below.
Twenty-eight above amino acids, shown in bold, are native PspA
amino acids from the NPB. The DNA sequence of the PCR product with
LIC ends (in italics) before using T4 DNA polymerase to
exonucleolytically digest the ends was:
TABLE-US-00028 [SEQ ID NO: 40]
GACGACGACAAGATGGAGAAGTCAGCAGATCAACAAGCTGAAGAAGACTA
TGCTCGTAGATCAGAAGAAGAATATAATCGCTTGACCCAACAGCAACCGT
AAACCGGGCTTCTCCTC
TABLE-US-00029 Calculated Molecular Weight = 34983.93 Estimated pI
= 5.58 Amino Acid Composition: No. Percent Non-polar: A 18 5.88 V
10 3.27 L 30 9.80 I 14 4.58 P 16 5.23 M 12 3.92 F 10 3.27 W 4 1.31
Polar: G 25 8.17 S 18 5.88 T 11 3.59 C 4 1.31 Y 16 5.23 N 5 1.63 Q
11 3.59 Acidic: D 26 8.50 E 24 7.84 Basic: K 25 8.17 R 14 4.58 H 13
4.25
Example 2
Mouse Immunization Studies
[0110] The recombinant proteins PAC001, PAC003, and MB001 were used
to immunize mice. MB001 contains SM-1 plus 1/2 of PAC001, including
the non-pro block. Mice were immunized subcutaneously three times
at ten-day intervals with alum as an adjuvant. Controls were
immunized with alum alone, or with alum plus recombinant protein
(fusion partner as a control that the process was not causing a
non-specific immunity). The reason for using a fusion partner in
this case is to facilitate purification and to be sure that the
peptides were large enough to have adequate immunogenicity.
Subsequently, the mice were challenged with a lethal dose of live
S. pneumoniae 3JYP2670 delivered intravenously by tail vein
injection. The challenge strain has a PspA that was non-cognate
with any of the three recombinant proteins and thus tested for
cross-protection. The times until the mice became moribund were
recorded, as reflected in FIG. 4 and FIG. 7. For each of the three
immunogens tested, protective responses were elicited as judged by
increased survival times.
[0111] Referring to FIG. 4, the results for MB001 were different
statistically from the alum control at P=0.021. The results for
PAC001 were different statistically from the alum control at
P=0.0006. For PAC003, the results were not quite significant
(0.0537) from alum, but three of the five mice survived versus two
of the fourteen for the alum control. The difference may become
more significant if more mice are used. The results with the fusion
partner were consistent with those obtained by alum, although the
number of mice tested was much smaller. It was clear from this
experiment that the proline-rich sequence of PspA can elicit
protection.
[0112] The recombinant protein from pCD42_NPB (GST_NPB) was made to
test whether this portion of the PspA and PspC peptides can elicit
protective antibodies in animal model systems and in humans. If
this activity is found, further studies will indicate how broad the
protection is (i.e. can it protect against strains with different
PspAs) Additionally, for example, whether antibodies can bind
encapsulated cells, whether antibodies can increase complement
deposition, and whether antibodies bind only growing cells are then
investigated.
[0113] Because the proline-rich region, whether it contains a
non-pro-block, is not part of the coiled-coil sequence, as
predicted in FIGS. 5A-C, and should not elicit any antibody
cross-reactive with epitopes in coiled-coil protein sequences
including those in myosin.
Example 3
Antibody Binding Studies
[0114] The monoclonal antibody designated KL67 was determined to
bind specifically with the non-proline block of the proline-rich
region embedded within some PspA and PspC proteins. Peptides of
PspA and KL67 specificity are shown in the stained gel and
corresponding Western blot depicted in FIG. 8.
[0115] The antibody binding of KL67 (anti-PspA-NPB) to
Streptococcus pneumoniae strains, as detected by flow cytometry
analysis using FITC-conjugated to mouse IgG, is depicted in FIG. 9.
Antibody was diluted 1/3 and shown is the average of three
experiments. Values on the vertical scale are "times greater than
control", is the quotient of fluorescence of the labeled bacteria
divided by the fluorescence of bacteria labelled with second
antibody only. The strains are given in legend and include D39
wildtype strain and mutants that are lacking expression of PspA and
PspC or capsule as indicated. The first three strains are
encapsulated. In strain D39, both the PspA gene and the PspC gene
have proline-rich regions that contain the NPB recognized by mAb
KL67.
[0116] Another study of the antibody binding of KL67 to
Streptococcus pneumoniae strains, as detected by flow cytometry
analysis using FITC-conjugated to mouse IgG, is summarized in FIG.
10. Antibody was diluted 1/3 and shown is the average of three
experiments. Values on the vertical scale are "times greater than
control", is the quotient of fluorescence of the labeled bacteria
divided by the fluorescence of bacteria labelled with second
antibody only. The strains are given in legend and include WU2
wildtype strain and WU2 mutants that are lacking expression of PspA
and Hic or capsule as indicated. The first three strains are
encapsulated. In the WU2 strain, only the PspA gene has a
proline-rich region that contains the NPB recognized by KL67. Hic
does not.
[0117] An additional study on the binding of KL67 pneumococcal
strains, as detected by flow cytometry analysis using
FITC-conjugated to mouse IgG, is summarized in FIG. 11. Antibody
was diluted 1/3 and shown is the average of three experiments.
Values on the vertical scale are "times greater than control", is
the quotient of fluorescence of the labeled bacteria divided by the
fluorescence of bacteria labelled with second antibody only. The
strains are given in legend and include TIGR4 wildtype strain and
mutants that are lacking expression of PspA, PspC, both or capsule
as indicated. The first five strains are encapsulated. In strain
TIGR4, only the PspC gene has a proline-rich region that contains
the NPB recognized by mAb KL67. PspA does not.
[0118] These studies indicate that mAb KL67 recognized specifically
the NPB region present in proline-rich regions of some PspA and
PspC molecules. The NPB in either the PspA or PspC context is
accessible to mAb KL67 antibody on live S. pneumoniae with capsule
2, capsule 3, and capsule 4 serotypes. Additionally, the NBP on
PspA is slightly more accessible to antibody in absence of PspC and
in the absence of capsule. Also, polyclonal sera raised against the
construct PAC001 binds the surface of pneumococcal cells.
Sequence CWU 1
1
401102PRTStreptococcus pneumoniae 1Asp Leu Lys Lys Ala Val Asn Glu
Pro Glu Lys Pro Ala Glu Glu Pro1 5 10 15Glu Asn Pro Ala Pro Ala Pro
Lys Pro Ala Pro Ala Pro Gln Pro Glu20 25 30Lys Pro Ala Pro Ala Pro
Ala Pro Lys Pro Glu Lys Ser Ala Asp Gln35 40 45Gln Ala Glu Glu Asp
Tyr Ala Arg Arg Ser Glu Glu Glu Tyr Asn Arg50 55 60Leu Thr Gln Gln
Gln Pro Pro Lys Ala Glu Lys Pro Ala Pro Ala Pro65 70 75 80Val Pro
Lys Pro Glu Gln Pro Ala Pro Ala Pro Lys Thr Gly Trp Gly85 90 95Gln
Glu Asn Gly Met Trp1002106PRTStreptococcus pneumoniae 2Asp Leu Lys
Lys Ala Val Asn Glu Pro Glu Thr Pro Ala Pro Ala Pro1 5 10 15Ala Pro
Ala Pro Ala Pro Ala Pro Thr Pro Glu Ala Pro Ala Pro Ala20 25 30Pro
Ala Pro Ala Pro Lys Pro Ala Pro Ala Pro Lys Pro Ala Pro Ala35 40
45Pro Lys Pro Ala Pro Ala Pro Lys Pro Ala Pro Ala Pro Lys Pro Ala50
55 60Pro Ala Pro Lys Pro Ala Pro Ala Pro Ala Pro Ala Pro Lys Pro
Glu65 70 75 80Lys Pro Ala Glu Lys Pro Ala Pro Ala Pro Lys Pro Glu
Thr Pro Lys85 90 95Thr Gly Trp Lys Gln Glu Asn Gly Met Trp100
105358PRTArtificialsynthetic 3Met Ala Lys Lys Ala Glu Leu Glu Lys
Thr Pro Glu Lys Pro Ala Glu1 5 10 15Glu Pro Glu Asn Pro Ala Pro Ala
Pro Gln Pro Glu Lys Ser Ala Asp20 25 30Gln Gln Ala Glu Glu Asp Tyr
Ala Arg Arg Ser Glu Glu Glu Tyr Asn35 40 45Arg Leu Thr Gln Gln Gln
Pro Pro Lys Ala50 55427PRTStreptococcus pneumoniae 4Glu Lys Ser Ala
Asp Gln Gln Ala Glu Glu Asp Tyr Ala Arg Arg Ser1 5 10 15Glu Glu Glu
Tyr Asn Arg Leu Thr Gln Gln Gln20 25510PRTStreptococcus pneumoniae
5Ala Ala Lys Lys Ala Glu Leu Glu Lys Thr1 5 10628PRTStreptococcus
pneumoniae 6Met Glu Lys Ser Ala Asp Gln Gln Ala Glu Glu Asp Tyr Ala
Arg Arg1 5 10 15Ser Glu Glu Glu Tyr Asn Arg Leu Thr Gln Gln Gln20
25728PRTStreptococcus pneumoniae 7Met Glu Lys Ser Val Asp Gln Gln
Ala Glu Glu Asp Tyr Ala Arg Arg1 5 10 15Ser Glu Glu Glu Tyr Asn Arg
Leu Thr Gln Gln Gln20 2588PRTStreptococcus pneumoniae 8Asp Leu Lys
Lys Ala Val Asn Glu1 5911PRTStreptococcus
pneumoniaeMISC_FEATURE(1)..(1)can be Lys or Gly 9Xaa Thr Gly Trp
Xaa Gln Glu Asn Gly Met Trp1 5 1010306DNAStreptococcus pneumoniae
10gaccttaaga aagcagttaa tgagccagaa aaaccagctg aagagcctga gaatccagct
60cctgcaccaa aaccagcgcc ggctcctcaa ccagaaaaac cagctccagc tcctgcacca
120aaaccagaga agtcagcaga tcaacaagct gaagaagact atgctcgtag
atcagaagaa 180gaatataacc gcttgactca acagcaaccg ccaaaagcag
aaaaaccagc tccagctcct 240gtaccaaaac cagagcaacc agctcccgca
ccaaaaacgg gctggggaca agaaaacggt 300atgtgg 30611318DNAStreptococcus
pneumoniae 11gaccttaaga aagcagttaa tgagccagaa actccagctc cggctccagc
cccagctcca 60gctccagctc caactccaga agccccagct ccagctccag ctccggctcc
taaaccagct 120ccggctccta aaccagctcc ggctcctaaa ccagctccgg
ctcctaaacc agctccggct 180cctaaaccag ctccggctcc taaaccagct
ccagctccag ctccggctcc taaaccagaa 240aagccagcag aaaaaccagc
tccagctcct aaaccagaaa ctccaaaaac aggctggaaa 300caagaaaacg gtatgtgg
31812174DNAStreptococcus pneumoniae 12atggctaaaa aagctgaatt
agaaaaaact ccagaaaaac cagctgaaga gcctgagaat 60ccagctccag caccacaacc
agagaagtca gcagatcaac aagctgaaga agactatgct 120cgtagatcag
aagaagaata taatcgcttg acccaacagc aaccgccaaa agca
1741384DNAStreptococcus pneumoniae 13gagaagtcag cagatcaaca
agctgaagaa gactatgctc gtagatcaga agaagaatat 60aatcgcttga cccaacagca
accg 841484DNAStreptococcus pneumoniae 14gagaagtcag cagatcaaca
agctgaagaa gactatgctc gtagatcaga agaagaatat 60aaccgcttga ctcaacagca
accg 841523PRTStreptococcus pneumoniae 15Asp Gln Gln Ala Glu Glu
Asp Tyr Ala Arg Arg Ser Glu Glu Glu Tyr1 5 10 15Asn Arg Leu Thr Gln
Gln Gln201640DNAStreptococcus pneumoniae 16gggagccatg gctgacctta
agaaagcagt taatgagcca 401736DNAStreptococcus pneumoniae
17ccgtcgacac cacataccgt tttcttgttt ccagcc 3618283PRTStreptococcus
pneumoniae 18Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe
Asp Thr Asp1 5 10 15Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe
Trp Ala Glu Trp20 25 30Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu
Asp Glu Ile Ala Asp35 40 45Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys
Leu Asn Ile Asp Gln Asn50 55 60Pro Gly Thr Ala Pro Lys Tyr Gly Ile
Arg Gly Ile Pro Thr Leu Leu65 70 75 80Leu Phe Lys Asn Gly Glu Val
Ala Ala Thr Lys Val Gly Ala Leu Ser85 90 95Lys Gly Gln Leu Lys Glu
Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly100 105 110Ser Gly His Met
His His His His His His Ser Ser Gly Leu Val Pro115 120 125Arg Gly
Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln130 135
140His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala
Met145 150 155 160Ala Asp Leu Lys Lys Ala Val Asn Glu Pro Glu Lys
Pro Ala Glu Glu165 170 175Pro Glu Asn Pro Ala Pro Ala Pro Lys Pro
Ala Pro Ala Pro Gln Pro180 185 190Glu Lys Pro Ala Pro Ala Pro Ala
Pro Lys Pro Glu Lys Ser Ala Asp195 200 205Gln Gln Ala Glu Glu Asp
Tyr Ala Arg Arg Ser Glu Glu Glu Tyr Asn210 215 220Arg Leu Thr Gln
Gln Gln Pro Pro Lys Ala Glu Lys Pro Ala Pro Ala225 230 235 240Pro
Val Pro Lys Pro Glu Gln Pro Ala Pro Ala Pro Lys Thr Gly Trp245 250
255Gly Gln Glu Asn Gly Met Trp Cys Arg Gln Ala Cys Gly Arg Thr
Arg260 265 270Ala Pro Pro Pro Pro Pro Leu Arg Ser Gly Cys275
28019327DNAStreptococcus pneumoniaemisc_feature(1)..(3)n is a, c,
g, or t 19nnnccatggc tgaccttaag aaagcagtta atgagccaga aaaaccagct
gaagagcctg 60agaatccagc tcctgcacca aaaccagcgc cggctcctca accagaaaaa
ccagctccag 120ctcctgcacc aaaaccagag aagtcagcag atcaacaagc
tgaagaagac tatgctcgta 180gatcagaaga agaatataac cgcttgactc
aacagcaacc gccaaaagca gaaaaaccag 240ctccagctcc tgtaccaaaa
ccagagcaac cagctcccgc accaaaaacg ggctggggac 300aagaaaacgg
tatgtggtgt cgacnnn 32720286PRTStreptococcus pneumoniae 20Met Ser
Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp1 5 10 15Val
Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp20 25
30Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp35
40 45Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln
Asn50 55 60Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr
Leu Leu65 70 75 80Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val
Gly Ala Leu Ser85 90 95Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn
Leu Ala Gly Ser Gly100 105 110Ser Gly His Met His His His His His
His Ser Ser Gly Leu Val Pro115 120 125Arg Gly Ser Gly Met Lys Glu
Thr Ala Ala Ala Lys Phe Glu Arg Gln130 135 140His Met Asp Ser Pro
Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met145 150 155 160Ala Asp
Leu Lys Lys Ala Val Asn Glu Pro Glu Thr Pro Ala Pro Ala165 170
175Pro Ala Pro Ala Pro Ala Pro Ala Pro Thr Pro Glu Ala Pro Ala
Pro180 185 190Ala Pro Ala Pro Ala Pro Lys Pro Ala Pro Ala Pro Lys
Pro Ala Pro195 200 205Ala Pro Lys Pro Ala Pro Ala Pro Lys Pro Ala
Pro Ala Pro Lys Pro210 215 220Ala Pro Ala Pro Lys Pro Ala Pro Ala
Pro Ala Pro Ala Pro Lys Pro225 230 235 240Glu Lys Pro Ala Glu Lys
Pro Ala Pro Ala Pro Lys Pro Glu Thr Pro245 250 255Lys Thr Gly Trp
Lys Gln Glu Asn Gly Met Trp Cys Arg Gln Ala Cys260 265 270Gly Arg
Thr Arg Ala Pro Pro Pro Pro Pro Leu Arg Ser Gly275 280
28521338DNAStreptococcus pneumoniaemisc_feature(1)..(3)n is a, c,
g, or t 21nnncatggct gaccttaaga aagcagttaa tgagccagaa actccagctc
cggctccagc 60cccagctcca gctccagctc caactccaga agccccagct ccagctccag
ctccggctcc 120taaaccagct ccggctccta aaccagctcc ggctcctaaa
ccagctccgg ctcctaaacc 180agctccggct cctaaaccag ctccggctcc
taaaccagct ccagctccag ctccggctcc 240taaaccagaa aagccagcag
aaaaaccagc tccagctcct aaaccagaaa ctccaaaaac 300aggctggaaa
caagaaaacg gtatgtggtg tcgacnnn 338229PRTStreptococcus pneumoniae
22Ala Lys Lys Ala Glu Leu Glu Lys Ala1 5239PRTStreptococcus
pneumoniae 23Ala Lys Lys Ala Glu Leu Glu Lys Thr1
5249PRTStreptococcus pneumoniae 24Thr Lys Lys Ala Glu Leu Glu Lys
Thr1 5259PRTStreptococcus pneumoniae 25Thr Lys Lys Ala Glu Leu Glu
Lys Thr1 52620PRTStreptococcus pneumoniae 26Pro Glu Lys Pro Ala Glu
Glu Pro Glu Asn Pro Ala Pro Ala Pro Gln1 5 10 15Pro Glu Lys
Ser20275PRTStreptococcus pneumoniae 27Pro Ala Pro Ala Pro1
5285PRTStreptococcus pneumoniae 28Pro Glu Lys Pro Ala1
52924DNAStreptococcus pneumoniae 29ggtacctgct tttggcggtt gctg
2430162DNAStreptococcus pneumoniae 30ctcgagatgg ctaaaaaagc
tgaattagaa aaaactccag aaaaaccagc tgaagagcct 60gagaatccag cttcagcacc
acaaccacaa caagctgaag aagactatgc tcgtagatca 120gaagaagaat
ataatcgctt gacccaagca ggaaaaacca gc 16231212DNAStreptococcus
pneumoniae 31gaattcacta gtgattctcg agatggctaa aaaagctgaa ttagaaaaaa
ctccagaaaa 60accagctgaa gagcctgaga atccagctcc agcaccacaa ccagagaagt
cagcagatca 120acaagctgaa gaagactatg ctcgtagatc agaagaagaa
tataatcgct tgacccaaca 180gcaaccgcca aaagcaggta ccaatcgaat tc
21232275PRTArtificialsynthetic 32Met Ser Asp Lys Ile Ile His Leu
Thr Asp Asp Ser Phe Asp Thr Asp1 5 10 15Val Leu Lys Ala Asp Gly Ala
Ile Leu Val Asp Phe Trp Ala Glu Trp20 25 30Cys Gly Pro Cys Lys Met
Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp35 40 45Glu Tyr Gln Gly Lys
Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn50 55 60Pro Gly Thr Ala
Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu65 70 75 80Leu Phe
Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser85 90 95Lys
Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly100 105
110Ser Gly His Met His His His His His His Ser Ser Gly Leu Val
Pro115 120 125Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe
Glu Arg Gln130 135 140His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp
Asp Asp Lys Ala Met145 150 155 160Ala Ile Ser Asp Pro Asn Ser Leu
Val Ile Leu Glu Met Ala Lys Lys165 170 175Ala Glu Leu Glu Lys Thr
Pro Glu Lys Pro Ala Glu Glu Pro Glu Asn180 185 190Pro Ala Pro Ala
Pro Gln Pro Glu Lys Ser Ala Asp Gln Gln Ala Glu195 200 205Glu Asp
Tyr Ala Arg Arg Ser Glu Glu Glu Tyr Asn Arg Leu Thr Gln210 215
220Gln Gln Pro Pro Lys Ala Gly Thr Asn Arg Ile Arg Ala Pro Ser
Thr225 230 235 240Ser Leu Arg Pro His Ser Ser Thr Thr Thr Thr Thr
Thr Glu Ile Arg245 250 255Leu Leu Thr Lys Pro Glu Arg Lys Leu Ser
Trp Leu Leu Pro Pro Leu260 265 270Ser Asn
Asn2753333DNAStreptococcus pneumoniae 33gacgacgaca agatggagaa
gtcagcagat caa 333439DNAStreptococcus pneumoniae 34gaggagaagc
ccggtttacc gttgctgttg agtcaagcg 3935306PRTArtificialsynthetic 35Met
Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro1 5 10
15Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu20
25 30Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu
Leu35 40 45Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp
Val Lys50 55 60Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp
Lys His Asn65 70 75 80Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu
Ile Ser Met Leu Glu85 90 95Gly Ala Val Leu Asp Ile Arg Tyr Gly Val
Ser Arg Ile Ala Tyr Ser100 105 110Lys Asp Phe Glu Thr Leu Lys Val
Asp Phe Leu Ser Lys Leu Pro Glu115 120 125Met Leu Lys Met Phe Glu
Asp Arg Leu Cys His Lys Thr Tyr Leu Asn130 135 140Gly Asp His Val
Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp145 150 155 160Val
Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu165 170
175Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys
Tyr180 185 190Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly
Trp Gln Ala195 200 205Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser
Asp Gly Ser Thr Ser210 215 220Gly Ser Gly His His His His His His
Ser Ala Gly Leu Val Pro Arg225 230 235 240Gly Ser Thr Ala Ile Gly
Met Lys Glu Thr Ala Ala Ala Lys Phe Glu245 250 255Arg Gln His Met
Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly260 265 270Asp Asp
Asp Asp Lys Met Glu Lys Ser Ala Asp Gln Gln Ala Glu Glu275 280
285Asp Tyr Ala Arg Arg Ser Glu Glu Glu Tyr Asn Arg Leu Thr Gln
Gln290 295 300Gln Pro30536117DNAStreptococcus pneumoniae
36gacgacgaca agatggagaa gtcagcagat caacaagctg aagaagacta tgctcgtaga
60tcagaagaag aatataatcg cttgacccaa cagcaaccgt aaaccgggct tctcctc
1173733DNAStreptococcus pneumoniae 37gacgacgaca agatggagaa
gtcagcagat caa 333839DNAStreptococcus pneumoniae 38gaggagaagc
ccggtttacc gttgctgttg agtcaagcg 3939306PRTStreptococcus pneumoniae
39Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro1
5 10 15Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His
Leu20 25 30Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe
Glu Leu35 40 45Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly
Asp Val Lys50 55 60Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala
Asp Lys His Asn65 70 75 80Met Leu Gly Gly Cys Pro Lys Glu Arg Ala
Glu Ile Ser Met Leu Glu85 90 95Gly Ala Val Leu Asp Ile Arg Tyr Gly
Val Ser Arg Ile Ala Tyr Ser100 105 110Lys Asp Phe Glu Thr Leu Lys
Val Asp Phe Leu Ser Lys Leu Pro Glu115 120 125Met Leu Lys Met Phe
Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn130 135 140Gly Asp His
Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp145 150 155
160Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys
Leu165 170 175Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile
Asp Lys Tyr180 185 190Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu
Gln Gly Trp Gln Ala195 200 205Thr Phe Gly Gly Gly Asp His Pro Pro
Lys Ser Asp Gly Ser Thr Ser210 215 220Gly Ser Gly His His His His
His His Ser Ala Gly Leu Val Pro Arg225 230 235 240Gly Ser Thr Ala
Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu245 250 255Arg Gln
His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly260 265
270Asp Asp Asp Asp Lys Met Glu Lys Ser Ala Asp Gln Gln Ala Glu
Glu275 280 285Asp Tyr Ala Arg Arg Ser Glu Glu Glu Tyr Asn Arg Leu
Thr Gln Gln290 295 300Gln Pro30540117DNAStreptococcus pneumoniae
40gacgacgaca agatggagaa gtcagcagat caacaagctg aagaagacta tgctcgtaga
60tcagaagaag aatataatcg cttgacccaa cagcaaccgt aaaccgggct tctcctc
117
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